Methodical Instruction for Students of the 1 Course Medical Faculty

 

LESSON ¹ 4 (PRACTICAL – 6 HOURS)

 

ThemeS: 1. Acid-base equilibrium in living organisms. Water product. Buffer solutions. Properties and preparing methods of buffer solutions.

2. Determination of the buffer solutions pH by colorimetric method.

3. Buffer capacity.

 

Aim: To be able to prepare buffer solutions, to calculate their pH and to determine capacity of buffer solutions.

 

Professional orientation of students:

Buffer systems are systems, which keep constant value pH. They are used in medical-chemical and sanitary-hygienic investigations because they can keep permanently values (7,36) in a blood. They can regulate concentration of hydrogen ion of biological liquid in living organisms. A lot of acidic compounds (it equivalently 10 liter 0,1 n HCl per one day) is produced in organism during habitability. Because hydrogen ion concentration affects living processes so profoundly, it is not surprising that regulation of pH has proven to be à universal and essential component of living organisms.

 

Methodology of Practical Class (900-1200).

I. Buffer solutions. To prepare and to study properties of buffer solutions.

Work 1. To prepare a acetic buffer solution.

Materials: 0,1 M of acetic acid solution, 0,1 M of sodium acetate solution

Protocol. In flask add: some mls of 0,1 M acetic acid solution and some mls of 0,1 M sodium acetate solution.

Calculation: pH buffer solution by the formula:       

; .

Also pH of this solution is determined by universal indicator paper.

Work 2.To study properties of pH buffer solution.

Materials: acetic buffer solution (which you preparing in work 1), 0,1mol/l solution of HCl, 0,1 mol/l solution NaOH

Protocol. To take 4 tubes. Add in three of them 5 mls of acetic buffer solution, but add 1 ml of acetic buffer solution in last tube.

Solution in first tube is used as control.

In Second tube add 0,5 mls HCl solution;

In Third tube add 0,5 mls NaOH solution;

In Fourth tube add 4 mls water;

In all tubes measured pH by universal indicator paper

To write result of yours observation in table:

 

II. Determination of the buffer solutions pH by colorimetric method.

Work 3. To prepare a buffer solution.

M a t e r i a l s: 0,1M of acetic acid solution, 0,1 M of sodium acetat solution

P r o t o c o l. To make 10 mls an acetic buffer. 

In tube add: 6 mls 0,1 M of acetic acid solution; 4 mls 0,1 M  of sodium acetat solution

C a l k u l a t i o n: pH buffer solution by the formula:       

Also pH of this solution is defined by universal indicator paper (approximate value of pH).

Work 4. To define pH buffer solution by colorimetric method.

If you know approximate value of pH, so you take indicator, which middle value transition range same as  pH of solution. For example, if the approximate value pH of solution equal 5 so you necessary take methyl red, as pH 5 is in the middle value transition range methyl red (4,2-6,2). After that in tube with buffer solution add 10 drops of this indicator solution and other same tube add 10 mls water. Both tubes take to middle foramens of comparator.

After that prepare a colour scale, because take eighteen tubes and place them in two series. In all tubes one of series add 10 mls of distilled water 1 drop of solution HCl (0,05 mol / l), but other series tubes add 10 mls of a distilled water and 1 drop NaOH (0,05 mol / l).

 In  tubes both series add some drops of indicator solution quantity drops of indicator solution, which you need add rite in table.

If you are prepared colour scale, so compare colour of solutions in tubes of a scale to colour of a buffered solution. If colour buffer solution such as colour in tubes of scale, so value pH of buffer are calculated by formula:

 

III. Buffer systems of blood. To determine capacity of buffer solution.

Work 5. To determine the acetic buffer solution of buffer capacity.

Materials: acetic buffer solution with pH0 = 4,25, 0,1mol/l solution of HCl, 0,1 mol/l solution NaOH, methyl orange solution, methyl red solution.

Protocol: add in 2 flasks 10 mls of acetic buffer solution;

a)  To determine the buffer capacity by acid

In ones of flask add 1 drop of the methyl orange.

This mixture are titrated by 0,1 mol/l hydrochloric acid solution until colour of methyl orange change from orange to red (pH1 = 3). To be calculated of capacity by formula:

mmole/l; where VHCl – quantity milliter of HCl solution, which you spend for titration.

b)   To determine the buffer capacity by base.

In second flask add 1 drop of the methyl red.

This mixture are titrated by 0,1 mol/l sodium hydroxide solution until colour of methyl red change from pink to orange (pH2 =5). To be calculated of capacity by formula:

 

mmole/l; where VNaOH – quantity milliter NaOH solution, which you spend for titration.

Work 6. To define buffering capacity of blood serum.

M a t e r i a l s: blood serum with pH0= 7,4, 0,1mol/l solution of HCl, 0,1 mol/l solution NaOH, methyl orange solution,  phenolphtalein solution.

P r o t o c o l. add in 2 flasks 10 mls blood serum;

a)To determine the buffer capacity by acid

 In one of them add 1 drop of the methyl orange

This mixture are titrated by 0,1 mol/l hydrochloric acid solution until colour of methyl orange change from orange to red (pH1 = 4). To be calculated of capacity by formula:

mmole/l; where VHCl – quantity milliter of HCl solution, which you spend for titration.

b) To determine the buffer capacity by base.

 In second flask add 1 drop of the phenolphtalein solution

This mixture are titrated by 0,1 mol/l sodium hydroxide solution until colour of phenolphtalein from colorless to pink (pH2=9). To be calculated of capacity by formula:

mmole/l; where VNaOH – quantity milliter NaOH solution, which you spend for titration.

Work 7. To determine of a drinking (tap) water buffer capacity.

Materials: water with pH0= 7.4, 0,1mol/l solution of HCl, 0,1 mol/l solution NaOH, methyl orange solution, phenolphtalein solution.

Protocol: add in 2 flasks 10 mls water;

a)  To determine the buffer capacity by acid.

In ones of flask add 1 drop of the methyl orange.

This mixture are titrated by 0,1 mol/l hydrochloric acid solution until colour change from orange to pink (pH1=3). To be calculated of capacity by formula:

mmole/l; where VHCl – quantity milliter HCl solution, which you spend for titration.

b)  To determine the buffer capacity by base.

In second flask add 1 drop of the phenolphtalein solution

This mixture are titrated by 0,1 mol/l sodium hydroxide solution  until colourof phenolphtalein change from colorless to crimson. (pH2=9)

The capacity by acid are calculated by formula:

mmole/l; where VNaOH – quantity milliter NaOH solution, which you spend for titration.

To writer result in the table:

Name of solution

Use on titration, ml

Buffering capacity

0,1 M HCl

0,1 M NaOH

By an acid

By an base

Acetic buffer solution

 

 

 

 

Blood serum

 

 

 

 

Drinking (tap) water

 

 

 

 

 

Individual Students Program.

I. Acid-base equilibrium in living organisms. Water product. Buffer solutions. Properties and preparing methods of buffer solutions.

1.   Acid-base equilibrium in living organisms.

2.   Water product. Buffer solutions.

3.   Properties and preparing methods of buffer solutions.

II. Determination of pH buffer solutions by colorimetric method.

1.   Determination of the buffer solutions pH by colorimetric method.

2.   Buffer capacity.

III. Buffer capacity.

1.                 Calculation pH of buffer solutions.

2.                 Buffer capacity.

3.                 Buffer systems of a blood, their composition and properties.

4.                 Feature of a buffer effect by each from buffer systems, their interaction with physiological functions of nephroses and mild.

 

Seminar discussion of theoretical issues (1230 – 1400 hour).

Test evaluation and situational tasks.

1. What is composition of Phosphate buffer solutions?

A.  HHb/Hb-;

B.  HHbO2/HbO2-;

C.  H2PO4-/HPO42-;

D.  H2CO3/HCO3-;

E.   NH3/NH4+

2. What is composition of hemoglobin buffer solution?

A. HHb/Hb-;

B. HHbO2/HbO2-;

C. H2PO4-/HPO42-;

D. H2CO3/HCO3-;

E.  NH3/NH4+

3. What is composition of oxyhemoglobin buffer solution?

A. HHb/Hb-;

B. HHbO2/HbO2-;

C. H2PO4-/HPO42-;

D. H2CO3/HCO3-;

E.  NH3/NH4+

 

Correct answers of test evaluations and situational tasks:

1.     C

2.     A,

3.     B.

 

Individual student work (1415-1500 hour)  are checked by solving situational tasks for each topic, answers in test evaluations and constructive questions (the instructor has tests & situational tasks).

 

Students must know:

-       properties of buffer solutions;

-       methods to define of buffer capacity of a buffer solution.

-       Composition of buffer solutions from the Brenstad-Loury theory;

-       Calculation pH of buffer solutions behind their composition;

-       Mechanism of a buffer effect at attachment of strong acids and alkalis.

 

Students should be able to:

-        make buffer solutions;

-       calculation pH of buffer solutions behind their composition;

-        determine of pH solution by colorimetric method.

-       to define buffer capacity of buffer solutions, Serum of a blood, a tap water.

 

References.

Basic:

1.     Atkins P.W. Physical chemistry. – New York. – 2004. – P.299-307.

2.     http://intranet.tdmu.edu.ua/Student’s facilities/Practical classes materials/Department of General Chemistry/medical chemistry/medical/1 course/English/04. Acid-base equilibrium in living organisms. Buffer solutions.

3.     Robert R Crichton; Frédéric Lallemand; Ioanna S M Psalti; Roberta J Ward. Biological inorganic chemistry: an introduction. Amsterdam [etc.] : Elsevier, 2010.

 

Additional:

1.     Lawrence D. Didona. Analytical chemistry. – 2002: New York. – P. 97– 118.

2.     John B.Russell. General chemistry. New York.2002. – P. 382-386

 

The methodical instruction has been worked out by: Associate Prof. Dmukhulska Ye. B.

 

 

 

 

Methodical instruction was discussed and adopted at the Department sitting

25.06.2013.  Minutes N 11

 

Methodical instruction was adopted and reviewed at the Department sitting

27.08.2013.  Minutes N 1